Clusterin is a widely distributed glycoprotein with a diverse set of biologic actions most prominent of which is its ability to promote cell interactions. A unique and defining feature of clusterin is its induction at times of tissue injury and remodelling. The function of clusterin in these settings is unclear. We hypothesize clusterin functions to initiate or maintain cell interactions, a common requirement of the states in which it is induced. We further hypothesize that the ability of clusterin to promote cell interactions protects against injury. The aim of this grant is to test these hypotheses. Hypothesis I. Clusterin promotes cell interactions. Preliminary data clearly documents the ability of exogenously applied clusterin to promote cell aggregation and adhesion. We will test the effects of induction or inhibition of clusterin expression on cell aggregation and adhesion using cell lines stably transfected with an inducible clusterin system in the sense or anti-sense orientation. To further test this hypothesis we will determine whether targeted deletion of both copies of the LLC-PK1 clusterin gene, by homologous recombination, inhibits the ability of these cells to adhere and spread. Developmental regulation of clusterin occurs during renal ontogeny. Using the metanephric organ culture model, we will test whether inhibition of clusterin, alters the more complex cell int~actions which characterize renal development. Hypothesis II. Clusterin has a protective function in the setting of renal injury. We will examine the ability of purified clusterin or endogenous overexpression of clusterin to promote cell interactions and protect membrane integrity in the setting of in vitro renal cell injury. Next, by targeting expression of clusterin to the cell membrane we will examine its ability to specifically protect this particular cellular domain. Finally the response to injury of homozygous mutant cells which have undergone targeted deletion of both copies of the clusterin gene will be examined. The ability of cells to interact may be an important determinant of their susceptibility to injury, thereby, accounting for the beneficial effects of clusterin. To test this hypothesis we will compare rates of O2 consumption, peroxide generation, intracellular pH and ATP content under basal and injury conditions in single cell suspensions or clusterin-induced cell aggregates. The last experiments using the in vivo model of ischemia-reperfusion injury in the rat, will examine the effects of clusterin administration or an anti-clusterin antibody on renal injury. The long-term aim of this grant is to further our understanding of the role of clusterin in the cell-cell interactions of the normal, developing and injured kidney. Such knowledge has the potential to lay the foundation for the development of new strategies for preventing or ameliorating renal or other organ injury.